Abstract

Background

Cellulose is highly recalcitrant and thus requires a specialized suite of enzymes
to solubilize it into fermentable sugars. In C. thermocellum, these extracellular enzymes are present as a highly active multi-component system
known as the cellulosome. This study explores the expression of a critical C. thermocellum cellulosomal component in T. saccharolyticum as a step toward creating a thermophilic bacterium capable of consolidated bioprocessing
by employing heterologously expressed cellulosomes.

Results

We developed an inducible promoter system based on the native T. saccharolyticum xynA promoter, which was shown to be induced by xylan and xylose. The promoter was used
to express the cellulosomal component cipA*, an engineered form of the wild-type cipA from C. thermocellum. Expression and localization to the supernatant were both verified for CipA*. When
a ΔcipA mutant C. thermocellum strain was cultured with a CipA*-expressing T. saccharolyticum strain, hydrolysis and fermentation of 10 grams per liter SigmaCell 101, a highly
crystalline cellulose, were observed. This trans-species complementation of a cipA deletion demonstrated the ability for CipA* to assemble a functional cellulosome.

Conclusion

This study is the first example of an engineered thermophile heterologously expressing
a structural component of a cellulosome. To achieve this goal we developed and tested
an inducible promoter for controlled expression in T. saccharolyticum as well as a synthetic cipA. In addition, we demonstrate a high degree of hydrolysis (up to 93%) on microcrystalline
cellulose.